Pubmed Dbd

Dengue virus neutralization by human immune sera: role ofenvelope protein domain III - reactive antibody

W.M. P. B. Wahala1, Annette A. Kraus1, Laura B. Haymore1, Mary Ann Accavitti-Loper2, andAravinda M. de Silva1,*1Department of Microbiology and Immunology, University of North Carolina School of Medicine,Chapel Hill, NC 275992Department of Medicine, University of Alabama at Birmingham, AL 35294

AbstractDengue viruses (DENV) are the etiological agents of dengue fever (DF) and dengue hemorrhagicfever (DHF). The DENV complex consists of four closely related viruses designated DENV serotypes1 through-4. Although infection with one serotype induces cross reactive antibody to all 4 serotypes,the long term protective antibody response is restricted to the serotype responsible for infection.Cross-reactive antibodies appear to enhance infection during a second infection with a differentserotype. The goal of the present study was to characterize the binding specificity and functionalproperties of human DENV immune sera. The study focused on domain III of the viral envelopeprotein (EDIII), as this region has a well characterized epitope that is recognized by stronglyneutralizing serotype-specific mouse monoclonal antibodies (Mabs). Our results demonstrate thatEDIII-reactive antibodies are present in primary and secondary DENV immune human sera. Humanantibodies bound to a serotype specific epitope on EDIII after primary infection and a serotype crossreactive epitope on EDIII after secondary infection. However, EDIII-binding antibodies constitutedonly a small fraction of the total antibody in immune sera binding to DENV. Studies with completeand EDIII antibody depleted human immune sera demonstrated that EDIII binding antibodies playa minor role in DENV neutralization. We propose that human antibodies directed to other epitopeson the virus are primarily responsible for DENV neutralization. Our results have implications forunderstanding protective immunity following natural DENV infection and for evaluating DENVvaccines.

IntroductionDengue viruses (DENVs) are emerging, mosquito-borne flaviviruses and the causative agentsof dengue fever (DF) and dengue hemorrhagic fever (DHF). The DENV complex consists offour serotypes designated DENV 1 through 4. A person infected with DENV developsantibodies that cross react with all four serotypes (Roehrig, 2003). However, the antibodiesonly provide long-term protection against the serotype responsible for the original infectionand people can be infected a second time with a different serotype (Halstead, 2002; Rothman,2004). Individuals experiencing secondary DEN infections face a greater risk of developingsevere disease (Halstead, 2002; Rothman, 2004). A leading theory to explain the greater risk

Aravinda M. de Silva, Ph.D., Department of Microbiology and Immunology, CB#7290 University of North Carolina School of Medicine,Chapel Hill, NC 27599, Tel: (919) 843-9964, Fax: (919) 962-8103, E-mail: [email protected]'s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptVirology. Author manuscript; available in PMC 2010 September 15.

Published in final edited form as:Virology. 2009 September 15; 392(1): 103–113. doi:10.1016/j.virol.2009.06.037.

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of severe disease with secondary DEN infection is that pre-existing cross reactive antibodiesbind to the virus and enhance infection of Fc-receptor bearing cells (Halstead, 2003). Despitethe fact that DEN vaccines are entering large scale clinical testing, we know remarkably littleabout the relationship between the binding properties of DEN antibodies in human immunesera and the functional outcome of these interactions.

The major target of flavivirus neutralizing antibody is the Envelope (E) protein, althoughmembrane protein (M) and non-structural protein 1 (NS1) antibodies have also been shown tobe protective (Roehrig, 2003; Schlesinger, Brandriss, and Walsh, 1987; Vázquez et al.,2002). E protein is responsible for viral attachment to host cells and the low pH fusion of viraland host cell membranes. The crystal structures of E of several flaviviruses have been solved(Modis et al., 2003; Modis et al., 2005; Nybakken et al., 2006; Rey et al., 1995). Individualsubunits of E consist of three beta-barrel domains designated E domains I (EDI), II (EDII) andIII (EDIII). Native E is a homodimer that lies flat on the surface of the viral membrane.

Our current understanding of the interactions between DENV and antibody is largely based onstudies with mouse monoclonal antibodies (Mabs). DENV neutralizing mouse Mabs have beenmapped to all three domains of E. In general, strongly neutralizing mouse Mabs are DENVserotype-specific and bind to an epitopes on EDIII that is unique to each serotype (Crill andRoehrig, 2001; Gromowski and Barrett, 2007; Lin et al., 1994; Lok et al., 2008; Roehrig, Bolin,and Kelly, 1998; Sukupolvi-Petty et al., 2007). A DENV type specific epitope on EDIII boundby strongly neutralizing Mabs has been mapped to 4 loops on the lateral face of EDIII(Gromowski and Barrett, 2007; Gromowski, Barrett, and Barrett, 2008; Sukupolvi-Petty et al.,2007). Investigators have also mapped flavivirus cross reactive epitopes on EDIII (Gromowski,Barrett, and Barrett, 2008; Sukupolvi-Petty et al., 2007). Unlike DENV type specific Mabs,cross reactive Mabs that bind to EDIII have moderate to weak neutralizing activity.

Despite the large body of work with mouse Mabs, remarkably little work has been done tocharacterize the binding properties of human DENV immune sera and to understand therelationship between human antibody binding and neutralization. Convalescent sera frompeople and horses naturally infected with West Nile virus (WNV), a related flavivirus, had lowlevels of EDIII-reactive antibody (Oliphant et al., 2007; Sanchez et al., 2007). In WNV immunesera, EDIII-binding antibodies were not primarily responsible for neutralization activity(Oliphant et al., 2007; Sanchez et al., 2007).

People who have recovered from DENV infections also develop EDIII-reactive antibodies(Beasley et al., 2004; Crill et al., 2009; Hapugoda et al., 2007; Holbrook, Shope, and Barrett,2004; Ludolfs et al., 2002); however, most human antibody appears to be directed towards aflavivirus-cross reactive epitope close to the fusion loop in EDII of DENV (Crill et al., 2009;Lai et al., 2008). To date, no studies have been done to directly test if EDIII-reactive antibodiesare primarily responsible for the neutralizing activity of human DENV immune sera. The goalof this study was to measure the level and specificity of EDIII-reactive antibodies in peoplewho have recovered from primary and secondary DENV infections and to determine thecontribution of EDIII-reactive antibodies to DENV neutralization.

Materials and MethodsViruses

DENV1 WestPac-74, DENV2 S-16803, DENV3 CH-53489, and DENV4 TVP-360, providedby Dr. Robert Putnak (Walter Reed Army Institute of Research, Silver Spring, MD) were usedin this study. Working virus stocks were obtained by inoculating C6/36 mosquito cells in tissueculture flasks and growing the virus for eight days at 28°C. Supernatants were harvested,clarified at 2500rpm for 5min, supplemented with 15% FBS and stored in aliquots at -80°C.

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Viral titers were determined by plaque assay on Vero-81 cells as previously described (Krauset al., 2007).

Immune sera and AntibodiesConvalescent DENV immune sera were obtained from volunteers who had experienced naturalDENV infections during previous travel abroad. The protocol for recruiting and collectingblood from people was approved by the Institutional Review Board of the University of NorthCarolina at Chapel Hill. Sera from 6 DENV-immune subjects were used in the present study.The properties of these sera are listed in Table 1. We also used eight DENV-reactive mouseMabs that bind to EDIII. Mab 3H5-1, obtained from Chemicon Co, CA, binds to EDIII ofDENV2 only (Gromowski and Barrett, 2007). Mabs 8A1 and 14A4, which bind to EDIII ofDENV3, were obtained from Dr. Robert Putnak (Walter Reed Army Institute of Research,Silver Spring, MD). Mab 2Q1899 and 9F16 which bind to EDIII of DENV2 were obtainedfrom United States Biological, Massachusetts. DENV3 EDIII reactive Mab 1H9 was obtainedfrom Dr John G Aaskov (Queensland University of Technology, Australia) (Serafin andAaskov, 2001) DENV cross reactive EDIII Mab 8A5 and 12C1 were developed in collaborationwith the South Eastern Regional Center for Excellence in Biodefence monoclonal antibodycore facility at the University of Alabama School at Birmingham.

Purification of DENV antigen for ELISADENV2 S-16803 and 3 CH53489 reference strains were grown in Vero-81 cells (ATCCCCL-81) at 37°C. The virus containing media was harvested 5-7 days after infection andcentrifuged to pellet cell debris. The clarified media was laid on top of a 20% sucrose (wt/vol)cushion and centrifuged (72,000g for 5 hrs) to pellet the virus. The virus pellet was allowed todissolve overnight in PBS before layering on a 10%- 40% iodixanol gradient and beingcentrifuged at 163,700 × g for 120 min. The virus-containing fractions were harvested. PBSwas added to the virus to dilute the iodixanol. The diluted solution was centrifuged (72,000 ×g for 5 h) to pellets the virus and remove the iodixanol. The virus pellet was resuspended inPBS and virus protein content was estimated by spectrometry. The virus was stored at -80°C.

Expression of the ectodomain of E protin (Es) from DENV3RNA extracted from strain CH53489 of DENV3 was resverse transcribed and PCR amplifiedto generate a PCR product containing nucleotides coding for the last 15 amino acids ofmembrane protein and the first 415 amino acids of E protein followed by a 6 histidine tag anda stop codon. This construct was missing the C-terminal amino acids responsible for membraneanchoring of E protein. The PCR product was cloned into pENTR TOPO vector (Invitrogen)and the BaculoDirect baculovirus Expression system (Invitrogen) was used to expressrecombinant protein according to manufacturer's instructions. Briefly, the Es gene wasrecombined with BaculoDirect Liner DNA to generate recombinant baculovirus DNA.pENTR/CAT plamsmid was used to produce recombinant baculovirus expressing thechloramphenicol acetyl transferase (CAT) protein, which was later used as a negative control.Sf9 insect cells were transfected with recombinant baculovirus DNA. P1,P2 and P3recombinant virus stocks were generated according to the manufacturers instruction andexpression of protein was confirmed using western blot with mouse Mab 4G2 ( for Es) or antiCAT antibodies. P3 baculovirus stocks were used to express Es and CAT proteins.

Expression and purification of DENV EDIIIRNA was extracted from supernatants of cells infected with DENV2 or 3 using QIAmp ViralRNA mini Kit (Qiagen). The nucleotide sequences encoding for EDIII of DENV2 (297-399AA) and DENV3 (295- 398 AA) were reverse transcribed and PCR amplified. The PCRproducts were cloned into pMAL c2X vector (NEB) to generate recombinant EDIII (MBP-

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EDIII) that is fused to maltose binding protein (MBP) at the N terminus according to themanufacture's instructions. MBP-EDIII from DENV2 and DENV3 were expressed inEscherichia coli DH5α (Invitrogen) and purified using amylose resin affinity chromatography(NEB).

Detection of dengue reactive antibody in human immune sera by ELISAELISA plates were also coated with 75ng/ per well of purified DENV2 or 3 and the flaviviruscross reactive Mab 4G2 was used to confirm equal binding of each virus to the plate. ELISAplates were coated with 200ng of MBP-EDIII from DENV2 or 3 per well. Rabbit anti MBPsera (NEB) was used to confirm equal binding of MBP-EDIII from both serotypes to ELISAplates. ELISA plates were coated using virus or protein recombinant protein antigen incarbonate buffer at pH 9.6 for 2 hrs at room temperature. The plates were washed 3 times inTris buffered saline with 0.2% Tween20 (TBST) and incubated with blocking buffer (Trisbuffered saline with 0.05% Tween20 containing 3% skim milk and 2% normal goat serum) at37°C for 1 hr. After washing the plates twice with TBST, human immune serum diluted inblocking buffer was added to each well and incubated at 37°C for 1 hr. Following 3 washeswith TBST, alkaline phosphatase-conjugated goat antihuman IgG (Fc-specific) (Sigma) wasadded to each well for 1 hour at 37°C. After 3 washes with TBST, p-nitrophenyl phosphatesubstrate (Sigma) was added to each well and the reaction was allowed to develop for 15minutes before recording optical density at 405nm on a spectrophotometer. In ELISAs withmouse Mabs, the protocol was the same except that alkaline phosphatase-conjugated goat anti-mouse IgG was used as a secondary antibody. To compare binding to DENV2 and DENV3antigens, we normalized the data by using the serum sample # 24 (secondary dengue) that gavethe highest OD with each antigen (DENV2, DENV3, DENV2 EDIII and DENV3 EDIII). Foreach antigen the maximum OD obtained with serum #24 was defined as an OD of 1. In figures1 and 4 the Y axis is referred to as relative OD to indicate that the data was normalized usingserum sample #24.

As the Es antigen bound poorly to ELISA plates, we used an antigen capture method to comparethe binding of whole virus and Es. Plates were coated with 200ng of Mab 8A5 in carbonatedbuffer at pH 9.6. This antibody binds to E protein from all 4 serotypes. The antibody coatedplates were washed and incubated with blocking buffer at 37°C for 1 hr. Next, sufficientDENV3 or Es antigen from DENV3 was added to saturate antigen binding to the antibodycoated plates. CAT protein antigen was used as a negative control. The plates were washedagain before incubating with serial dilutions of dengue immune human sera. The rest of theassay was performed as described above for the direct antigen coating ELISA.

Depletion of EDIII-reactive antibody in human immune seraPurified MBP-EDIII was dialyzed against 20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mMEDTA (column buffer) overnight at 4°C. MBP-EDIII (300 ug) was incubated with the amyloseresin (NEB) in column buffer containing 3% normal human serum (NHS) and incubatedovernight at 4°C. The resin was washed three times with column buffer and 3 more times withPBS to remove unbound MBP-EDIII. The resin was blocked with 5% NHS in PBS beforeincubating with 1.5mls of human DENV immune serum diluted at 1:10 in PBS for 4 hrs at 37°C. The amylose resin was pelleted and the EDIII antibody depleted human serum was collected.Depletion of EDIII-reactive antibodies was confirmed by ELISA with MBP-EDIII. In addition,each human immune serum sample was absorbed to an amylose resin with MBP alone. MBPabsorbed immune sera and MBP-EDIII absorbed NHS were used as negative controls insubsequent neutralization assays

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DENV Neutralization assaysDENV neutralizing antibodies was measured by plaque reduction neutralization test (PRNT)or a flow cytometry based neutralization assay. The PRNT was performed as previouslydescribed (Kraus et al., 2007). In brief, Vero-81 cells were seeded into 24 well-plates and grownuntil 80% confluent. Serially diluted sera were mixed with 30 plaque forming units (PFU) ofvirus and incubated for 1 hr at 37°C. The virus/serum mix was added to the Vero cells andincubated with a nutrient overlay medium (Opti-MEM® with 1% methylcellulose and 10%FBS) for four days at 37°C. The cells were fixed and stained for viral antigen with monoclonalantibody 4G2 as previously described (Kraus et al., 2007). The percentage of neutralizationwas defined as reduction in the number of foci in the test sera compared to the number of fociin the control wells with normal human serum. The 50% neutralization titers were determinedby nonlinear dose-response regression analysis (Prism Package, GraphPad Software, Inc., SanDiego, CA). Flow cytometry based neutralization assays were performed in 96-well plates withthe U937 human monocytic cell line transfected with DC-SIGN as previously described (Krauset al., 2007). In brief, immune sera were serially diluted and incubated with sufficient virus toinfect 10 to 15% of the cells in the well. The virus/serum mixture was incubated for 1hr at 37°C and then added to the cells for 1hr at 37°C. The cells were washed to remove unbound virusand fresh media was added before incubating cells for 24hrs at 37°C. Cells were fixed,permeabilized and stained with DENV Mabs 4G2 or 2H2, both of which bind to all fourserotypes (Kraus et al., 2007). Cells were analyzed with a FACScan flow cytometer (BectonDickinson) to identify infected cells. The 50% neutralization titers were determined bynonlinear dose-response regression analysis ((Prism Package, GraphPad Software, Inc., SanDiego, CA).

ResultsDengue immune human sera were obtained by collecting blood samples from volunteers whomight have been infected during foreign travel. Of 35 subjects enrolled in the study, 17 hadantibodies that neutralized one or more DENV serotypes. The neutralization patterns of the 17immune subjects were consistent with past exposures to DENV1 only (one subject), DENV2only (four subjects), DENV3 only (four subjects) and secondary DENV infections (eightsubjects). These sera were also tested for DENV neutralizing antibody by the Centers forDisease Control (CDC) in Fort Collins, CO and the Laboratory of Infectious Diseases, NationalInstitute of Allergy and Infectious Diseases (NIAID), Bethesda, Maryland. The CDC andNIAID laboratories reached the same conclusions as we did about the past infection history ofthese subjects (unpublished data from Drs Robert Lanciotti, CDC and Steve Whitehead, NIH).For the current study we selected 6 sera representing 2 subjects each who had recovered fromprimary DENV2, primary DENV3 and secondary DENV infections. The DENV neutralizationtiters and the most likely year and place of infection of these subjects are listed in Table 1.

DENV Binding Antibodies in Human Immune SeraExperiments were performed to measure the binding properties of antibodies in the 6 selectedimmune sera to purified DENV2 and 3. The immune sera were tested at four fold dilutionsstarting at 1:50. Antibodies in human DENV immune sera cross reacted with both serotypesindicating that the dominant antibodies after primary and secondary infection are serotypecross-reactive (Figure 1). End point virus binding titers were calculated for the 6 sera (Table2). As expected, subjects with secondary infections had higher titers than subjects with primaryinfections (Table 2). These results indicate that an ELISA with whole virus as antigen mainlydetects serotype cross reactive antibodies and the assay is not predictive of the neutralizationproperties of the serum sample or past infections history of the subject.

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The DENV particle is made up of envelope (E), membrane (M) and capsid (C) proteins. As Eprotein is the main target of neutralizing antibody, experiments were done to compare theantibody response to E protein and whole virions. As full length E protein alone is not secretedout of cells, we expressed the soluble ectodomain of E (Es) from DENV3 to be used as anantigen. We used immune serum samples # 003 and 011 from primary DENV3 cases and #009 and 024 from secondary cases and compared binding to DENV3 and Es from DENV3.Antibodies in human immune sera bound well to both Es and virus particles, but greater bindingwas observed with virus particles compared to Es (Figure 2). These results demonstrate thatalthough the ectodomain of E is a dominant target of antibody, virions contain epitopes thatare absent in recombinant Es.

Purification and Characterization of Recombinant DENV Envelope Protein Domain III (EDIII)Studies with mouse Mabs have demonstrated that most DENV serotype-specific antibodiesbind to EDIII (Crill and Roehrig, 2001; Gromowski and Barrett, 2007; Lin et al., 1994; Lok etal., 2008; Roehrig, Bolin, and Kelly, 1998; Sukupolvi-Petty et al., 2007). When using wholevirus antigen in an ELISA, the cross-reactive antibodies in human immune sera are likely todominate and mask signal originating from serotype-specific antibodies. To develop an assayfor measuring serotype-specific antibody, recombinant EDIII was expressed as a MBP fusionprotein in E. coli (Figure 3). Previous studied have demonstrated that EDIII expressed aloneor as a MBP fusion protein is folded correctly and displays antibody epitopes present on thevirion (Maillard et al., 2008; Volk et al., 2004; Volk et al., 2007; Yu et al., 2004). To confirmthat recombinant DENV2 and 3 MBP-EDIII fusion proteins produced in our laboratory werecorrectly folded, binding assays were performed with eight mouse Mabs that bind to EDIII ofDENV2 and/or 3. Mabs 3H5-1, 9F16 and 2Q1899 are antibodies that bind to serotype-specificepitopes on the lateral ridge of DENV2 (Gromowski and Barrett, 2007; Henchal et al., 1985;Sukupolvi-Petty et al., 2007). As predicted, all three antibodies bound to MBP-EDIII fromDENV2 but not DENV3 (Table 3). We used mouse Mabs 8A1, 14A4 and 1H9 which areserotype-specific neutralizing antibodies that bind to EDIII from DENV3 only (Serafin andAaskov, 2001) (unpublished data, Putnak, Wahala and de Silva). Mab 8A1 and 1H9 bind tothe lateral ridge of EDIII from DENV3, whereas the 14A4 epitope on DENV3 EDIII has notbeen mapped yet. Mabs 8A1, 14A4 and 1H9 bound to MBP-EDIII from DENV3 but notDENV2 (Table 3). We also used two neutralizing Mabs that bind to a serotype cross reactiveepitopes in EDIII and these two antibodies bound to both recombinant proteins (Table 3). Thus,the type specific and cross reactive neutralizing epitopes on EDIII of DENV are preserved inthe recombinant proteins used in the current study.

EDIII-reactive antibodies in human DENV immune seraAfter confirming that the recombinant EDIII–MBP fusion proteins expressed appropriateserotype-specific and cross reactive epitopes, the antigens were used to detect EDIII-reactiveantibody in our panel of human DENV immune sera. Each immune serum was tested in four-fold dilutions starting at 1:12.5. At high concentrations of serum, antibodies in the two subjectswith evidence of past primary DENV2 infections bound to EDIII from DENV2 better thanEDIII from DENV3 (Figure 4A and B). Similarly, antibodies in the two subjects with serotype-specific neutralizing antibody to DENV3 bound to EDIII from DENV3 better than EDIII fromDENV2 (Figure 4C and D). The two subjects with evidence of past secondary DENV infectionshad antibodies that bound equally well to both antigens (Figure 4E and F). These resultsdemonstrate that EDIII-reactive antibodies that developed after primary infection were specificto the serotype responsible for infection. The EDIII end point binding titers also displayedserotype-specificity after primary infection (Table 2). In the case of secondary serum samples,the EDIII end point titers were similar for both antigens (Table 2).

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We compared the amount of antibody in immune sera directed to the whole virus versus EDIII.To measure relative amounts of available EDIII epitopes on viral and recombinant proteinantigens used in the binding assays, end point titers were calculated using MAbs 8A5 and 12C1(Table 3), which bind to a conserved DENV complex epitope on EDIII. The endpoint titerswere 5-10 times higher for recombinant EDIII compared to virus (Table 2). This result wasexpected because the DENV complex epitope on EDIII has been mapped to the A-B loop,which is poorly exposed on the intact virus but not on recombinant EDIII (Sukupolvi-Petty etal., 2007). Despite the superior binding of 8A5 and 12C1 to recombinant EDIII, human immunesera bound poorly to recombinant EDIII compared to the virus antigen (Table 2). After primaryinfections the serotype-specific EDIII-reactive antibodies ranged from 0.1 to 8.1% of total virusreactive antibodies (Table 2). Following secondary infections the EDIII-reactive antibodiesaccounted for 0.2 to 4.0 % of total virus reactive antibodies. These results indicate that subjectswho have recovered from DENV infections have low levels of EDIII-reactive antibody and,in the case of primary infections, antibodies are directed to serotype-specific epitope(s) onEDIII.

Role of EDIII-reactive Antibodies in DENV NeutralizationExperiments were performed to determine the contribution of EDIII-reactive antibodies inhuman immune sera to DENV neutralization. The immune sera were depleted of EDIII bindingantibodies by incubating the serum samples with MBP-EDIII bound to an amylose resin. Theprimary DENV2 and DENV3 immune sera were incubated with MBP-EDIII from DENV2 orDENV3, respectively. The secondary sera were treated with MBP-EDIII from DENV2(Sample 009) or DENV3 (Sample 024). As depicted in Figure 5, incubation with recombinantMBP-EDIII removed most of the EDIII-reactive antibody. The treatment specifically removedEDIII-reactive antibodies as sera treated with MBP alone were indistinguishable fromuntreated immune sera (Figure 5). Interestingly, when the secondary sera were depleted usingMBP-EDIII from one serotype, most EDIII reactivity to the second serotype was also lost (datanot shown) indicating that in secondary immune sera the antibodies are mainly directed againsta cross-reactive epitope on EDIII.

As the recombinant EDIII used in above studies was expressed as a MBP fusion protein, it wasconceivable that some human antibody epitopes in EDIII were altered or masked by the fusionpartner. To determine if MBP fusion partner altered important epitopes on EDIII, bindingassays were performed with purified DENV2 EDIII without MBP (kindly provided by Dr.Michael Diamond, Washington University, St. Louis). Dengue immune sera absorbed withrecombinant DENV2 EDIIII-MBP were tested for the presence of antibodies that bound toEDIII without MBP. As depicted in Figure 6, immune sera absorbed with DENV2 MBP-EDIIIprotein failed to bind recombinant DENV2 EDIII without MBP indicating that the MBP fusionpartner does not alter or mask the main human antibody epitopes on EDIII.

Next, untreated and EDIII antibody depleted sera were tested in the flow cytometry basedDENV neutralization assay with U937 cells expressing DC-SIGN. Figure 7 depictsneutralization curves for primary DENV2, primary DENV3 and secondary DENV immunesera. Serum samples with or without EDIII-reactive antibodies showed similar neutralizationpatterns (Figure 7). The 50% neutralization titers were ∼ 10-15% lower for serum samplesdepleted with EDIII compared to the MBP treated sera (Table 4). These results indicate thatEDIII-reactive antibodies make a minor contribution to the total neutralizing capacity of humanDENV immune sera (Table 4).

As it was conceivable that EDIII antibodies might play an important role in DENVneutralization in some cell types but not others, some of the experiments with U937 cellsexpressing DC-SIGN (Table 4) were repeated with Vero cells. We selected EDIII antibodydepleted serum sample # 003 (primary DENV3 immune) and # 009 (secondary DENV

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immune) and performed neutralization assays with Vero cells. In the case of sample #003, theneutralization titers for DENV3 were similar for untreated and EDIII antibody depleted serum(50% neutralization titers of 74 and 88 respectively). Similarly, for sample #009, theneutralization titers for DENV2 were similar (50% neutralization titers of 895 and 858respectively) for untreated and EDIII antibody depleted sera. These results demonstrate thatEDIII reactive antibodies were not required to neutralize DENV infection of U937 cells andVero cells.

DiscussionDespite many publications on interactions between DENV and antibody, surprisingly fewstudies have been published that on how the binding properties of human antibodies relate toDENV neutralization. The goal of the current study was to characterize the specificity andfunctionality of antibodies in DENV immune human sera. Here we have demonstrated thatEDIII reactive antibodies are present in human immune sera. The EDIII antibodies mainlyrecognized a type specific epitopes after primary infection and a cross reactive epitope aftersecondary infection. EDIII binding antibodies were a minor component of the total antibodiesin immune sera binding to DENV. Recently Crill and co-workers used DENV2 virus likeparticles to measure epitopes specific human antibody responses and they also observed lowlevels of EDIII binding antibodies in human sera (Crill et al., 2009). Our results demonstratethat EDIII binding antibodies make only a minor contribution to the total neutralizing capacityof human immune sera. Thus, the EDIII neutralizing epitopes that have been the focus of muchrecent work (Gromowski and Barrett, 2007; Gromowski, Barrett, and Barrett, 2008; Sukupolvi-Petty et al., 2007) were not the target of most neutralizing antibody in primary and secondaryDENV immune sera.

Several investigators have reported the presence of EDIII-reactive antibodies following naturalinfection of people and animals with flaviviruses (Beasley et al., 2004; Hapugoda et al.,2007; Holbrook, Shope, and Barrett, 2004; Ludolfs et al., 2002; Sanchez et al., 2007). In humanWNV immune sera EDIII-reactive antibodies were present, although at low levels comparedto the total antibody binding to virus (Oliphant et al., 2007). Furthermore, investigators haveshown that EDIII-reactive antibodies are specific for the infecting virus, unlike antibodiesagainst the whole virus particle, which are highly cross-reactive (Beasley et al., 2004;Hapugoda et al., 2007; Holbrook, Shope, and Barrett, 2004; Ludolfs et al., 2002; Sanchez etal., 2007). Our data reported here indicate that DENV cross reactive antibodies dominate inwhole virus binding assays. Our data demonstrate that in subjects who have recovered fromprimary DENV infection, EDIII-reactive antibodies were mainly directed to an epitope specificfor the serotype responsible for infection, whereas in secondary cases EDIII-reactive antibodiesbound to a DENV cross-reactive epitope. Recent studies with mouse Mabs have mapped thelocation of both cross reactive and serotype specific epitopes on DENV EDIII (Gromowskiand Barrett, 2007; Gromowski, Barrett, and Barrett, 2008; Sukupolvi-Petty et al., 2007).Although it is reasonable to speculate that the cross reactive and serotype specific epitopesdefined by mouse Mabs are also targets of the human antibody response measured here, furtherstudies are needed to confirm this.

Currently, the primary serological assay to identify the DENV serotype responsible for aprimary infection is the neutralization test, which is a laborious and time consuming assay. Ourresults indicate that a simple ELISA with recombinant EDIII as antigen can be used to identifythe DENV serotype responsible for primary infection. Our results also demonstrate that thespecificity of EDIII-reactive antibodies is preserved in both early (within first year, data notshown) and late (>4 years after infection) convalescent primary sera. In secondary infections,this assay is unlikely to predict responsible serotypes as the response is directed to cross reactiveepitope(s) on EDIII. Ludolfs and colleagues also reported similar results using recombinant

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EDIII in an immunoblot assay with human immune sera (Ludolfs et al., 2002). Further studiesare needed to evaluate the utility of recombinant EDIII as an antigen for identifying DENVsresponsible for primary infection. In the case of secondary infections, studies need to addressif the EDIII-reactive antibodies simply cross-react with the serotypes responsible for theprimary and secondary infections or if they also cross- react with serotypes not responsible forinfection.

Studies with mouse Mabs have led to the identification and mapping of a serotype-specificepitope on the lateral ridge of EDIII of several flaviviruses including DENV (Gromowski andBarrett, 2007; Nybakken et al., 2005; Roehrig, 2003; Sukupolvi-Petty et al., 2007). An implicit,but untested assumption has been that antibodies directed to this epitope must play a role inserotype-specific neutralization following natural human infection. Our results demonstratethat DENV-specific EDIII-reactive antibodies play a minor role in neutralization observed withhuman sera. Studies with immune sera from people and horses naturally infected with WNVhave also revealed a variable role for EDIII-reactive antibodies in viral neutralization (Oliphantet al., 2007; Sanchez et al., 2007). In some cases, EDIII-reactive antibody depletion led to adecrease in WNV neutralization whereas in other cases no significant change was observed(Oliphant et al., 2007; Sanchez et al., 2007). Our results do not rule out the possibility of interdomain epitopes involving EDIII making important contributions to neutralization as theseepitopes would not be present in the recombinant EDIII proteins used in the current study. Weconclude that antibodies directed to inter domain epitopes, epitopes on EDI or II of E proteinand, possibly M protein, are mainly responsible for the neutralizing activity of human immunesera.

One potential concern is that the recombinant EDIII MBP fusion proteins used here may beimproperly folded and not display important epitopes present on EDIII in the virus particle.Our studies indicated that the recombinant proteins were correctly folded. Eight EDIII-reactive,neutralizing mouse Mabs bound with appropriate specificity to the recombinant EDIII fromDENV2 or 3 used here indicating that the main type specific and cross reactive neutralizingepitopes described in the literature were present on our recombinant EDIII proteins(Gromowski and Barrett, 2007; Gromowski, Barrett, and Barrett, 2008; Henchal et al., 1985;Serafin and Aaskov, 2001; Sukupolvi-Petty et al., 2007). Other groups have performedstructure studies with EDIII expressed alone or as a MBP-fusion protein and demonstrated thatthe E. coli expressed protein has a structure similar to EDIII in its native form (Volk et al.,2004; Volk et al., 2007; Yu et al., 2004). We also demonstrated here fusing EDIII to MBP didnot mask or alter the main human antibody epitopes in EDIII because human sera absorbedwith MBP-EDIII failed to bind to EDIII without the MBP fusion portion as well.

In summary, our results indicate that EDIII-reactive antibodies are of minor importance inneutralizing DENV by human DENV immune sera. We propose that the major cross-reactiveand serotype-specific neutralizing epitopes targeted by human immune sera are inter-domainepitopes (Goncalvez et al., 2004) and/or located outside EDIII. Currently live attenuated DENVvaccines are being tested in human clinical trials (Edelman, 2007). It is reasonable to assumethat the protective antibodies induced by these vaccines will be similar to protective antibodiesinduced after a natural DENV infection. We predict that the quality and quantity of antibodiesagainst EDIII will not determine the efficacy of live attenuated DENV vaccines. As analternative approach to live attenuated vaccines, several groups have focused on developingrecombinant EDIII vaccines (Babu et al., 2008; Bernardo et al., 2008; Etemad et al., 2008).Given the low levels of EDIII-reactive antibodies detected here in human immune sera, cautionis urged in proceeding with EDIII based platforms. We currently do not understand why peopledevelop low levels of EDIII-reactive antibody after natural infection. The human immunesystem may recognize and react to epitopes on EDI and EDII better than to EDIII. However,with appropriate adjuvants and recombinant protein constructs, it might be possible to stimulate

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an effective immune response directed to relevant epitopes on EDIII as well. Such vaccinesare likely to neutralize DENV by a mechanism that is different from neutralization observedafter natural infection. The topic of flavivirus-antibody interactions has been dominated bystudies to identify and characterize epitopes on EDIII. We hope the results reported here willstimulate more work to characterize epitopes on EDI and EDII and their role in DENVneutralization.

AcknowledgmentsWe are thankful to Nicholas Olivarez for excellent technical assistance. We also thank Drs. Robert Lanciotti (CDC,Fort Collins, CO) and Stephen Whitehead (NIAID, Bethesda, MD) for testing human sera for DENV neutralizingantibody. We are especially indebted to Dr. Robert Putnak ((Walter Reed Army Institute of Research, Silver Spring,MD) and Dr. John Aaskov (Queensland University of Technology, Australia) for providing us virus strains andantibodies. We thank Drs. Michael Diamond and James Brian (Washington University, St. Louis, MO) for providingpurified recombinant EDIII from DENV2. These studies were supported by a targeted research grant (DR-11B) fromthe Pediatric Dengue Vaccine Initiative (PDVI), which is an initiative funded by the Bill and Melinda Gates Foundationas well as NIH grant #U54 AI057157 from Southeastern Regional Center of Excellence for Emerging Infections andBiodefense

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Figure 1.Binding of human immune sera to purified DENV2 and 3. The binding of antibodies inconvalescent sera from patients who have recovered from primary DENV2 infections (A, B),primary DENV3 infections (C, D) and secondary DENV infections (E, F) to purified DENV2(solid lines) or DENV3 (dashed line) was analyzed by ELISA. The data points represent meanvalues and the error bars represent the standard error of the mean. The data shows one of tworepresentative experiments.

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Figure 2.Binding of human immune sera to purified DENV3 and the ectodomain of E protein. Tocompare antibody binding to whole virions and E protein, ELISA plates were coated withpurified DENV3 or the ectodomain of E protein (Es) from DENV3. As a negative control,plates were coated with chloramphenicol acetyl transferase (CAT) protein. Sera from patientswho have recovered from primary DENV3 infections (03 and 011) and secondary DENVinfections (09, and 024) were used to measure virus and E protein specific antibody responses.The data shows one of two representative experiments.

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Figure 3.Purification and characterization of recombinant MBP-EDIII fusion proteins from DENV2 and3. The recombinant protein expressed in E. coli and purified by amylase affinitychromatography. Lanes 1 and 3 depict the DENV2 and DENV3 MBP-EDIII fusion proteinsin E. coli lysates. Lanes 2 and 4 depict the purified protein obtained after amylase affinitychromatography. The arrow indicated the band corresponding to the 53kD MBP-EDIII fusionprotein.

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Figure 4.Binding of human immune sera to MBP-EDIII from DENV2 or 3. Convalescent sera from 2people each who had recovered from primary DENV2 (A, B), primary DENV3 (C, D) andsecondary DENV (E, F) infections were tested for binding to recombinant MBP-EDIII fromDENV2 (solid lines) or DENV3 (dashed line). The data points represent the mean values andthe error bars represent the standard error of the mean. The data are from one of tworepresentative experiments.

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Figure 5.Depletion of EDIII-reactive antibody from human immune sera. Immune sera were fromsubjects who had have recovered from primary DENV2 (Panel A), primary DENV3 (Panel B)or secondary DENV (Panel C) infections. The sera were absorbed using MBP alone orrecombinant MBP-EDIII-fusion protein from DENV2 (sera # 001, 009 and 013) or DENV3(sera #003, 011 and 024).

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Figure 6.Binding of EDIII antibody depleted dengue immune sera to DENV2 EDIII without a MBPfusion partner. Dengue immune sera (# 13, and #24) were absorbed using MBP-EDIII or MBPalone. The absorbed sera were tested for binding to DENV2 EDIII expressed without a MBPfusion partner. The MBP-EDIII absorbed sera failed to bind to EDIII alone indicating thatEDIII with or without a MBP expressed similar antibody epitopes.

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Figure 7.DENV neutralization by human immune sera depleted of EDIII binding antibodies.Convalescent sera from 2 people each who had have recovered from primary DENV2 (A, B),primary DENV3 (C, D) and secondary DENV (E, F) infections were depleted of EDIII bindingantibody and tested for DENV neutralization at different dilutions. Neutralizing antibody wasmeasured using U937 cells expressing DC-SIGN and flow cytometry. Neutralization curvesare depicted for untreated (open circles), MBP treated (open triangles) and EDIII antibodydepleted (dark squares) sera. The data are from one of two representative experiments.

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